Proper functioning of the immune system requires elaborate network of negative regulatory mechanisms to modulate the strength of activation signal or to disengage the immune response after successful completion of a defense mounted against pathogens. One of the recent additions to this negative regulatory network is hematopoietic progenitor kinase 1 (HPK1), a member of the KHS subfamily of Ste20 serine/threonine kinases. We disrupted the HPK1 locus and found that mice genetically lacking HPK1 resist the growth of Lewis Lung Carcinoma (LLC). The presence of tumor infiltrating lymphocytes (TILs) and T cell transfer into T cell-deficient mice revealed that tumor rejection is T cell-mediated. HPK1-/- T cells produce more IL-2 than wild type counter part. They are hyperresponsive to activation signals and are resistant to apoptosis and immunosuppressive factors produced by tumor cells. In this proposal, we will evaluate the two know properties of HPK1 - its ability to negatively regulate TCR signaling pathway and its ability to induce apoptosis. Targeted disruption of HPK1 locus result in enhanced anti-cancer immunity, but it could not implicate which of these two HPK1-regulated functions contribute to this phenotype. We will determine whether the role of HPK1 as a kinase serves the primary function of inhibiting T cell activation, and upon losing that function, T cells can respond to cancer in a more robust manner. Alternatively, we will determine whether HPK1's role as a caspase-3 substrate and a regulator of apoptosis plays a more significant role in anti-cancer immunity. In aim 1, we will use HPK1-/- T cells that have been reconstituted with kinase defective mutant of HPK1 or with caspase-3 cleavage site mutated form. These reconstituted T cells will be adoptively transferred to into tumor-bearing RAG2-/- mice to evaluate their ability to mount anti-cancer immune response. In aim 2, we will identify downstream targets of HPK1 and screen for small molecule(s) that would inhibit the specific HPK1- mediated signal transduction pathway involved in anti-cancer immunity. We will also identify proteins that bind to HPK1-C fragment and determine which of these proteins are involved in regulating the anti-cancer immunity. Not only will these studies reveal the importance of each HPK1-regulated function in anti-cancer immunity, but the identification of HPK1 kinase substrates and isolation of small molecule compound(s) that could inhibit HPK1 kinase activity will greatly advance our understanding of the role HPK1 play in T cell biology. Successful identification of small molecule inhibitor of HPK1 could one day develop into anti-cancer drugs that stimulate T cells to attack cancer cells more robustly. PUBLIC HEALTH RELEVANCE: HPK1 is a molecule that control the activity of anti-cancer cells known as "T cells". Take away HPK1 from T cells will enable T cells to fight cancer more effectively. In this proposal, we described a series of experiments that may shed light as to how HPK1 is accomplishing this task.